AIM:To optimize conditions of DHPLC and analyze theeffectiveness of various DNA polymerases on DHPLCresolution,and evaluate the sensitivity of DHPLC in themutation screening of mitochondrial DNA(mtDNA).METHODS:Two fragments of 16s gene of mitochondrial DNA(one of them F2 is a mutant fragment)and an A3243Gmutated fragment were used to analyze the UV detectionlimit and determine the minimum percentage of mutant PCRproducts for DHPLC and evaluate effects of DNApolymerases on resolution of DHPLC.Under the optimalconditions,we analyzed the mtDNA mutations from muscletissues of mitochondrial encephalomyopathy with lacticacidosis and stroke-like episodes(MELAS)and screenedblindly for variances in D-loop region of rntDNA from humangastric tumor specimen.RESULTS:Ten A3243G variants were detected in 12 cases ofMELAS,no alterations were detected in controls and theseresults were consistent with the results obtained by analysisof RFLP with Apal.We also identified 26 D-loop variances in46 cases of human gastric cancer tissues and 38 alterationsin 13 gastric cancer cell lines.The mutation of mtDNA at 80 ngPCR products containing a minimum of 5% mutant sequencescould be detected by using DHPLC with UV detector.Moreover,Ampii-Taq Gold polymerase was equally as good asthe proofreading DNA polyrnerase(e.g.,Pfu)in eliminatingthe false positive produced by Taq DNA polymerases.CONCLUSION:DHPLC is a powerful,rapid and sensitivemutation screening method for mtDNA.Proofreading DNApolymerase is more suitable for DHPLC analysis than Taqpolymerase. AIM: To optimize conditions of DHPLC and analyze the effectiveness of various DNA polymerases on DHPLC resolution, and evaluate the sensitivity of DHPLC in themutation screening of mitochondrial DNA (mtDNA). METHODS: Two fragments of 16s gene of mitochondrial DNA (one of them F2 is a mutant fragment) and an A3243Gmutated fragment were used to analyze the minimum percentage of mutant PCR products for DHPLC and evaluate effects of DNApolymerases on resolution of DHPLC. Under the optimal conditions, mouse analysis the mtDNA mutations from muscletissues of mitochondrial encephalomyopathy with lacticacidosis and stroke-like episodes (MELAS) and screenedblindly for variances in D-loop region of rntDNA from humangastric tumor specimens. RESULTS: Ten A3243G variants were detected in 12 cases of MELAS, no alterations were detected in controls and these results were consistent with the results by analysis of RFLP with Apal. We also identify 26 D-loop variances in 46 cases of human gastric cancer tissues and 38 alterations in 13 gastric cancer cell lines. The mutation of mtDNA at 80 ngPCR products containing a minimum of 5% mutant sequences detected by using DHPLC with UV detector. Moreover, Ampii-Taq Gold Polymerase was equally as good asthe proofreading DNA polyrnerase (eg, Pfu) in in the false positive produced by Taq DNA polymerases. CONCLUSION: DHPLC is a powerful, rapid and sensitive screening screening method for mtDNA. Proofreading DNApolymerase is more suitable for DHPLC analysis than Taqpolymerase.